THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine (PC) is a widespread response of diverse cells to stimulation with a wide range of agonists and thereby implicated in a broad spectrum of physiological processes including metabolic regulation, inflammation, secretion, mitogenesis and the immune response. The lipid product of this PC-specific PLD, phosphatidic acid (PA), is generally believed to constitute the signal output of this reaction. In vitro, PA modulates the activity of a varied group of regulatory proteins. Metabolism of PA generates biologically active molecules including diacylglcerol, arachidonic acid and lyso-phosphatidic acid. Certain PLD activities are stimulated by members of ADP-ribosylation factor (ARF) and Rbo families of monomeric GTP-binding proteins including RhoA, Rac and cdc42, suggesting that PLD is a mediator of the protein trafficking and mitogenic signaling processes respectively controlled by these proteins. The research described in this proposal focuses on two recently identified mammalian PLD enzymes termed PLD1 and PLD2. The proteins will be expressed recombinantly from cloned cDNAs, purified and their catalytic properties, substrate selectivities, regulation by ARF and Rho family G-proteins, phospholipid cofactors and protein kinases examined in detail. The involvement of PLD1 and PLD2 in receptor-regulated PC hydrolysis will be examined by transient expression in cells and examination of PLD activity in response to stimulation of endogenous or co-expressed receptors. Concurrently, we will explore the mechanisms coupling receptors to activation of PLD1 and PLD2 and define the role of these enzymes in agonist-promoted alterations in phospholipid metabolism and mitogenic signaling that accompany PLD-catalyzed PC hydrolysis. We will test the hypothesis that PLD1 and/or PLD2 is involved in the process by which ARF regulates the binding of the heptameric coatomer complex to Golgi-derived membrane vesicles.